Odontology DOI 10.1007/s10266-013-0140-3

ORIGINAL ARTICLE

TGF-b in dentin matrix extract induces osteoclastogenesis in vitro Wannakorn Sriarj • Kazuhiro Aoki • Keiichi Ohya • Mariko Takahashi • Yuzo Takagi Hitoyata Shimokawa

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Received: 13 August 2013 / Accepted: 20 November 2013 Ó The Society of The Nippon Dental University 2013

Abstract Previously, we have demonstrated that the extracellular matrix from dentin affects osteoclastic activity in co-culture between osteoclast and osteoblast-rich fraction from mouse marrow cells. In the present study, we aimed to investigate the mechanisms of dentin matrix extract-induced osteoclastogenesis in mouse bone marrow macrophages (BMMs). Dentin proteins were extracted from bovine incisor root dentin using 0.6 M HCl. BMMs were cultured in a-MEM containing macrophage colonystimulating factor/receptor activator of nuclear factor kappa-B ligand in the presence or absence of dentin matrix extract. Tartrate-resistant acid phosphatase (TRAP)-positive cell number, total TRAP activity, and the mRNA

W. Sriarj (&) Department of Pediatric Dentistry, Faculty of Dentistry, Chulalongkorn University, 34, Henri-Dunant Road, Pathumwan, Bangkok 10330, Thailand e-mail: [email protected] K. Aoki  K. Ohya  M. Takahashi Pharmacology, Department of Bio-matrix, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan e-mail: [email protected] K. Ohya e-mail: [email protected] M. Takahashi e-mail: [email protected] Y. Takagi  H. Shimokawa Pediatric Dentistry, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan e-mail: [email protected] H. Shimokawa e-mail: [email protected]

levels of osteoclast-related genes, assayed by real-time RTPCR, were determined as markers of osteoclastogenesis. A neutralizing antibody against transforming growth factorb1 (TGF-b1), SB431542, a TGF-b receptor inhibitor, and ELISA were used to determine the role of TGF-b1. We observed increases in TRAP-positive cell number, TRAP activity, and the mRNA levels of osteoclast-related genes of BMMs cultured with dentin extract. The use of a neutralizing antibody against TGF-b1 or SB431542 inhibited the inductive effect of dentin extract, suggesting TGF-b1 involvement. The addition of exogenous TGF-b1, but not bone morphogenic protein-2, also increased osteoclastogenesis, corresponding to the ELISA determination of TGF-b1 in the dentin extract. In conclusion, our results indicate that proteins from dentin matrix have an inductive effect in osteoclastogenesis, which is mediated, in part, by TGF-b1. Keywords Dentin organic matrix  Mouse bone marrow macrophage  Osteoclast  Osteoclastogenesis  TGF-b

Introduction Tooth root resorption occurs under both physiological and pathological conditions by the action of odontoclasts, which have similar functions and cytological characteristics to osteoclasts [1–3]. In primary teeth, the root dentin can undergo either physiologic or pathologic resorption, while only pathologic root resorption is found in permanent teeth. In bone, constant physiologic turnover occurs under the influence of hormonal control and functional forces. While the mechanisms of bone remodeling are well investigated, less is known about the factors regulating root resorption.

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During tooth development, the odontoblasts secrete an unmineralized extracellular matrix (ECM) layer, the socalled predentin. Predentin becomes mineralized when hydroxyapatite crystals are deposited within the collagen matrix of the predentin [4]. Collagenous proteins comprise over 90 % of the dentin ECM and the remainder are collectively referred to as the non-collagenous proteins (NCPs) [5]. The dentin NCPs, including dentin phosphoprotein (DPP) and dentin sialoprotein (DSP), which are predominantly expressed by odontoblasts [6, 7], dentin matrix protein 1 (Dmp1), bone sialoprotein (BSP), and osteocalcin [4] are involved in the regulation of mineralization. In addition to ECM proteins, growth factors such as TGF-b, IGF, BMPs, and FGFs are also secreted by odontoblasts and are trapped in dentin during mineralization [8– 10]. However, the physiological roles of these growth factors in dentin remain unclear. ECM proteins and growth factors have been shown to be released from the dentin matrix as a consequence of osteoclast action during root resorption [11]. These molecules may regulate resorption by modulating the activity of the cells that take part in this process [12, 13]. ECM proteins have been demonstrated to facilitate osteoclast activity [14–16]. Moreover, the involvement of growth factors within the dentin, such as IGF-I, BMP-2, and FGF2, on osteoclast formation and resorption activity has been demonstrated [17–19]. However, the role of TGF-b1, the most abundant growth factor found in dentin, remains unresolved. TGF-b1 is a multifunctional growth factor that has been reported to regulate some aspects of tooth development [20, 21]. It has also been shown that TGF-b1 released from dentin after injury participates in tissue repair [22–25]. In addition, TGF-b1 also plays a crucial role in osteoclast formation and activity [26–28]. However, the effect of TGF-b1 on osteoclasts was reported to be dependent on the culture system used. TGF-b1 inhibited osteoclastogenesis when added to co-cultures of osteoblasts and hematopoietic cells, but enhanced the osteoclastogenesis of RANKL- or TNF-a-stimulated hematopoietic cells [26, 27, 29]. We have previously shown that dentin matrix extract (DME) could inhibit osteoclast activity [30]. However, the detailed mechanism of DME on osteoclastogenesis is still unclear. In our previous study, DME was added to cocultured osteoclast precursors and osteoblasts; therefore, it was difficult to distinguish the effect of DME on osteoblasts or osteoclast precursors. In the present study, we aimed to examine the influence of DME on osteoclast precursors to clarify the direct effect of DME on osteoclasts. The activity of TGF-b1 from dentin matrix on osteoclastogenesis was also investigated.

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Materials and methods Animals and reagents Seven-week-old male ICR mice were purchased from Sunkyo Labo Service (Saitama, Japan). All animal procedures were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University, Tokyo, Japan. Benzamidine hydrochloride n-hydrate/benzylsulfonyl fluoride (PMSF) was purchased from Wako Pure Chemical (Osaka, Japan). Alpha-amino-n-caproic acid was obtained from Sigma (St. Louis, MO, USA). TGF-b1 and SB431542 were purchased from PeproTech (London, UK) and TOCRIS bioscience (Ellisville, MO, USA) respectively. AntiTGF-b1 antibody and BMP-2 were purchased from R&D Systems (Minneapolis, MN, USA). Preparation of protein extracts from bovine dentin Protein was extracted from bovine permanent incisor root dentin as previously described [31]. Briefly, the cleaned roots were cut into small pieces with a nipper, then rinsed several times with phosphate-buffered saline (PBS) and then washed in PBS containing protease inhibitors (2.5 mM benzamidine HCL, 50 mM a-amino-n-caproic acid, and 0.3 mM PMSF) by stirring overnight at 4 °C. Protein was extracted from the dentin shards with 0.6 M HCL (35 volume/weight of dentin) for 4 days at 4 °C. After centrifugation at 10,0009g for 30 min, the extract, which contained protease inhibitors, was dialyzed extensively against distilled water and lyophilized. The lyophilized extract was resuspended in culture medium at a concentration of 1 mg/ ml, centrifuged at 12,0009g for 10 min, and the supernatant was collected for use in the assays. In vitro osteoclast formation Bone marrow (BM) cells obtained from the femora and tibiae of 7-week-old male ICR mice were plated on 10-cm diameter culture dishes and cultured for 24 h in a-MEM (Sigma-Aldrich, St. Louis, MO, USA), 10 % FBS (Moregate, Biotech, Bulimba QLD, Australia) containing 5 ng/ ml recombinant human M-CSF (R&D systems) in 5 % CO2 at 37 °C. Non-adherent BM cells were harvested and used as osteoclast progenitor cells. The non-adherent BM cells (105 cells) were seeded in 96-well dishes in a-MEM, with 10 % FBS, 20 ng/ml recombinant human M-CSF, and 50 ng/ml soluble RANKL (Wako Pure Chemical) [32]. Dentin extract at concentrations of 0, 5, 10, 20, or 50 lg/ml was added to the cultures, which were incubated for 5 days at 37 °C in 5 % CO2. In other experiments, TGF-b1 was added at a concentration of 1 or 10 ng/ml or BMP-2 was

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added at a concentration of 10 or 100 ng/ml. Media with or without factors was replaced on day 3. On day 5, the cells were fixed with 4 % formalin and stained for TRAP, a marker enzyme of osteoclasts, as previously described [33]. Osteoclast number was determined by counting the number of TRAP-positive cells with three or more nuclei.

determine the total protein concentration of the cell lysates. TRAP activity was calculated as lmol liberated p-nitrophenol/well.

Measurement of TRAP Enzymatic activity

Osteoclast progenitor cells were incubated with or without dentin extract or TGF-b1 or BMP-2 for 5 days. Total RNA was isolated using a Nucleospin II kit, according to the manufacturer’s directions (BD Biosciences Clontech, Palo Alto, CA). The mRNA was reverse transcribed using a First-strand cDNA synthesis Kit (Roche Diagnostics). Real-time RT-PCR was performed on a LightCyclerÒ 480 system (Roche Diagnostics) using SYBR green for the detection of PCR products. The following genes were analyzed: cathepsin K, TRAP, CTR, v-ATPase, RANK, Integrin b3, TRAF 6, and NFATc1. The PCR primer sequences used are shown in Table 1. Target gene expression was normalized to GAPDH levels.

A rapid microplate colorimetric assay with modifications was performed to determine TRAP activity [34]. Briefly, osteoclast progenitor cells treated as above were lysed in 1 % NP-40 lysis buffer containing protease inhibitor (Complete-EDTA free, Roche Diagnostics, Indianapolis, IN, USA). The cell lysates were incubated with p-nitrophenyl phosphate (pNPP; 6.7 mM) as substrate in 100 mM sodium acetate, 50 mM sodium tartrate buffer (pH 5.9) at 37 °C for 60 min. The reaction was stopped by the addition of 3 M NaOH; the absorbance was read at 405 nm. A BCA Protein Assay Kit (Pierce, Rockford, IL, USA) was used to

Table 1 Real-time RT-PCR primers

Gene

Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR)

Forward primer Reverse primer

Product (base pairs)

Sequence references

50 -TGTATAACGCCACGGCAAA-30

195

X94444

101

NM007588

99

NM008084

153

AF239169

140

AF019046

89

NM009424

155

NM016780

151

BC029644

161

AB022322

Cathepsin K Forward Reverse CTR

50 -GGTTCACATTATCACGGTCACA-30

Forward

50 -TCAGGAACCACGGAATCCTC-30

Reverse

50 -ACATTCAAGCGGATGCGTCT-30

GAPDH Forward

50 -CTCCCACTCTTCCACCTTCG-30

Reverse

50 -TTGCTGTAGCCGTATTCATT-30

NFATc1 Forward Reverse

50 -CAACGCCCTGACCACCGATAG-30 0

5 -GGGAAGTCAGAAGTGGGTGGA-3

0

RANK Forward

50 -GGAAGCAAATCTATACCCCCA-30

Reverse

50 -GAGTCAGTTCTGCTCGGA-30

TRAF6 Forward

50 -GCGCTGTGAAGTCTCTACCC-30

Reverse

50 -GACGCTACACCCCCGCATCA-30

b3-integrin Forward

50 -TGACTCGGACTGGACTGGCTA-30

Reverse

50 -ACTTCTCACAGGTGTCTCCAT-30

TRAP Forward

50 -TACCTGTGTGGACATGACC-30

Reverse

50 -CAGATCCATAGTGAAACCGC-30

v-ATPase Forward

50 -TCCAACACAGCCTCCTACTT-30

Reverse

50 -ACAGCAAAGGCAGCAAAC-30

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Quantitative immunoassay for TGF-b1 (ELISA) To measure the TGF-b1 content, freeze-dried dentin extract was resuspended in PBS and the supernatant was used after centrifugation at 12,0009g at 4 °C for 10 min. The TGF-b1 content of the dentin extract was measured using a Quantikine ELISA Kit (R&D systems), according to the manufacturer protocol. The absorbance was read at 450 nm.

Fig. 1 Dentin extract enhanced TRAP activity in RANKLstimulated osteoclast progenitor cell culture. Dentin extract was added in a range of concentrations and cultured for 5 days. TRAP staining and TRAP-positive cell number are illustrated (a). TRAP activity is expressed as nmol/lg protein/ min (b). Data are expressed as mean ± SD (n = 3). *p \ 0.05 versus control (0 lg/ml). The morphology of cells changed to stellate cell shape (arrow) when cultured with 50 lg/ml of dentin extract (c). The bar indicates 50 lm

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Inhibition of TGF-b1 using a neutralizing anti-TGF-b1 antibody A monoclonal anti-TGF-b1 antibody was used to study the inhibition of TGF-b1 bioactivity. As previously described, osteoclasts were formed in a-MEM, 10 % FBS, containing dentin extract, with or without 5 lg/ml neutralizing anti-TGFb1 antibody. The concentration of the anti-TGF-b1 antibody used was according to the manufacturer’s instructions.

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Inhibition of TGF-b using SB431542 To evaluate the effect of TGF-b in the dentin extract on osteoclastogenesis, SB431542, a TGF-b type I receptor inhibitor, was added to the culture. As described above, osteoclasts were formed in a-MEM, with 10 % FBS, containing dentin extract, with or without 1 or 5 lM SB431542 [35, 36]. SB431542 was dissolved at a concentration of 10 mM in DMSO as a stock solution. The same volume of DMSO without inhibitor was added in the

control cultures. The concentration of SB431542 used (1 or 5 lM) was based on our preliminary study.

Statistical analysis All data were expressed as mean ± SD. The statistical significance between groups was analyzed using Student’s t test or one-way ANOVA followed by Tukey’s post hoc test. Statistical significance was set at p \ 0.05.

Fig. 2 Osteoclast progenitor cells were cultured for 5 days with or without 20 lg/ml of dentin extract (dentin). Dentin extract up-regulated the mRNA levels of Cathepsin K, TRAP, CTR, v-ATPase, RANK, Integrin b3, TRAF6, and NFATc1 as shown in a–h, respectively. Data are expressed as mean ± SD (n = 3). *p \ 0.05 versus control (Cont)

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Results In the present study, the influence of DME on osteoclast formation and activity was examined. BMMs were cultured in the presence of various concentrations of DME, ranging from 0 to 50 lg/ml. As shown in Fig. 1, after 5 days, DME could increase the number of TRAP-positive cells and total TRAP activity in a dose-dependent manner. Real-time RTPCR was used to assay the mRNA expression of target genes. Figure 2 shows the significant inductive effect of DME on the expression of the osteoclast-related genes, cathepsin K, TRAP, CTR, v-ATPase, RANK, and integrin b3. Interestingly, the up-regulation of NFATc1 and TRAF6, which are key regulators of osteoclastogenesis, was also observed. Because some BMMs cultured with 50 lg/ml of DME exhibited an irregular stellate cell shape (Fig. 1c), we decided to use DME at a concentration of 20 lg/ml in further experiments. TGF-b1, the most abundant growth factor in the dentin matrix, has been reported to be involved in multiple stages of dental hard tissue formation [20–22, 27]. We hypothesized that TGF-b1 might play a role in DME-induced osteoclastogenesis. To investigate the activity of TGF-b1, a TGF-b receptor inhibitor or a TGF-b1 neutralizing antibody was added in combination with DME and the results are presented in Fig. 3. The addition of anti-TGF-b1 antibody (Fig. 3a) or 1 or 5 lM of SB431542 (Fig. 3b) could attenuate the inductive effect of DME as indicated by the significant reduction of TRAP activity. No significant effect was observed in cultured using control IgG compared with the control (data not shown). To further confirm the effect of TGF-b1, exogenous TGFb1 was added to the BMM culture media. Exogenous BMP-2 was used as a control because BMP belongs to the TGF-b superfamily and is also entrapped in dentin during mineralization. These two factors have been reported to influence osteoclast development [17, 26, 27]. The results showed that both DME and TGF-b1 significantly increased TRAP activity, whereas BMP-2 did not, as compared with control (Fig. 4a). In addition, exogenous TGF-b1 could induce the expression of osteoclast-related genes including TRAF6 and NFATc1 (Fig. 4b) to levels similar to those induced by DME. The presence of TGF-b1 in DME was also determined by ELISA. As shown in Fig. 4c, dentin matrix contained up to 57.94 ± 10.58 ng of TGF-b1 per mg of dentin (n = 3).

Discussion To our knowledge, no studies have directly demonstrated the effect of DME on osteoclastogenesis. Here, we report that DME from root dentin induced osteoclastogenesis in a RANKL-stimulated bone marrow cell culture system.

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Fig. 3 Anti-TGF-b1 antibody and SB431542 inhibited the effect of dentin extract (dentin) on osteoclast formation. Addition of 5 lg/ml of anti-TGF-b1 antibody (a) or 1 and 5 lM of SB431542 (b) to the cultures attenuated the inductive effect of dentin matrix. Data are expressed as mean ± SD (n = 3). *p \ 0.05 compared with cells treated with dentin extract without anti-TGF-b1 antibody or SB431542

Osteoclasts are multinucleated giant cells, derived from the monocyte/macrophage lineage of hematopoietic stem cells in bone marrow. Osteoclasts play a crucial role in bone resorption. The regulation of osteoclast formation requires the function of two factors, RANKL and M-CSF [37, 38]. The binding of RANKL to its cognate receptor, receptor activator of nuclear factor kB (RANK) on the osteoclast surface, elicits signal transduction stimulating the expression of specific osteoclast gene markers [39], such as TRAP, cathepsin K, CTR, and integrin avb3. Many studies have reported the effects of various cytokines, including TGF-b1, in regulating osteoclastogenesis [29, 40, 41]. In the present study, the direct effect of DME on osteoclastogenesis in a BMM culture system was demonstrated. The up-regulation of TRAP activity and osteoclast related-genes indicated that DME could enhance osteoclast formation. The results were in agreement with reports showing direct role of TGF-b1 on osteoclastogenesis. TGFb1 has been shown to directly increase osteoclast formation in the absence of osteoblasts [29, 40, 41]. The inhibition of TRAP activity by anti-TGF-b1 antibody and SB431542 when used in conjunction with DME suggests that TGF-b1 entrapped in DME could promote osteoclast formation. In our previous study, DME was added to a co-culture of osteoclast progenitor cells and osteoblasts; therefore, it was

Odontology Fig. 4 Exogenous TGF-b1 induced osteoclast formation. BMMs were treated with dentin extract (dentin) or TGF-b1 or BMP-2. a Dentin and TGF-b1 showed an increase in TRAP activity, while BMP-2 did not. b The mRNA levels of osteoclast-related genes including TRAF6 and NFATc1 were induced by exogenous TGF-b1, similar to dentin extract. c The TGF-b1 content in dentin extract was determined by ELISA. Data are expressed as mean ± SD (n = 3). *p \ 0.05 versus control (Cont)

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not possible to separate the effect of DME on osteoclast progenitor cells from its effect on osteoblasts. It is interesting to note the differential effects of DME on the two culture systems. While a negative effect on osteoclastogenesis was observed in the co-culture system, an inductive effect was found when DME was used to stimulate osteoclast progenitor cells. The discrepancy between these two experiments raises the hypothesis that DME may have different effects on osteoclast progenitor cells compared with osteoblasts. Alternatively, the response of osteoclast precursors to DME may be different in the presence of osteoblasts. Indeed, DME may influence the osteoblast and promote the release of osteoclast inhibitory factor(s), resulting in the inhibitory effect observed in the co-culture [30]. In this study, the presence of TGF-b1 in DME was demonstrated using an ELISA analysis. In addition, using inhibition and stimulation approaches it was found that TGF-b1 could directly induce osteoclastogenesis. While the influence of TGF-b1 on osteoclast progenitors has been demonstrated in this paper, the role of TGF-b1 in the coculture between osteoblast and osteoclast is still unclear. Our results were in agreement with a report from Quinn et al. [29], who showed that TGF-b had differential effects on different osteoclast culture systems. During the process of dentin destruction, TGF-b1 released from exposed dentin might play a role in accelerating osteoclastogenesis, resulting in root resorption [12]. On the contrary, TGF-b1 released from damaged dentin in pulp tissue could influence the differentiation of odontoblast-like cells [24]. These reports suggested the dual function of TGF-b1 in both anabolic and catabolic reactions. It is possible that function of TGF-b is depended either on particular type of cells or on the balance between hard tissue-forming and hard tissue-resorbing cells, of which, the resorption process is either initiated or inhibited. Further study is required to clarify the notion. Beside TGF-b1, it has been reported other isoforms of TGF-b; TGF-b2, and TGF-b3 were also entrapped in the dentin matrix [8, 22] and could be extracted from dentin [22]. Moreover, BMP, especially BMP-2, is also found in dentin. It is possible that these growth factors can also influence the osteoclastogenesis [42, 43]; however, using exogenous growth factor our results showed that BMP-2 up to 100 ng/ ml has no effect on osteoclast formation. More experiments are needed to clarify the role of BMP-2 as well as TGF-b2 and TGF-b3 in dentin extract on osteoclastogenesis. The addition of recombinant TGF-b1 demonstrated the direct effect of TGF-b1 on osteoclast precursors. Osteoclasts have been shown to express TGF-b receptors; therefore, they can directly respond to the growth factor. The reduction in osteoclastogenesis we observed following the application of a TGF-b receptor inhibitor supports the

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direct effect of TGF-b1 on osteoclastogenesis. The signaling cascade involved in the response needs further investigation. Our findings demonstrated that DME and exogenous TGF-b1 could up-regulate the expression of osteoclastogenesis-related genes. It has been shown that TGF-b1 could induce the expression of RANK, which binds to RANKL on the osteoclast surface [40], TRAF6, one of the key signaling molecules in osteoclastogenesis [44], and NFATc1, which is an important regulator of osteoclastogenesis [45]. In agreement with those studies, the present study found a significant up-regulation of RANK, TRAF6, and NFATc1 when osteoclast precursor cells were treated with dentin extract or TGF-b1. These effects are likely partly mediated by the direct stimulation of TGF-b1 in DME on osteoclast precursor cells. However, in our study, we did not measure the effect of TGF-b1 on RANKL/MCSF/OPG expression. Although the results clearly showed the inductive effect of TGF-b1 on osteoclast related genes, it is difficult to identify whether the effect was direct or indirect via other intermediate molecules. In conclusion, our study demonstrates the inductive effect of dentin extract on osteoclast precursor cells, similar to that of TGF-b1. This effect could be partly abrogated by an antiTGF-b1 antibody or a TGF-b receptor inhibitor. This indicates that TGF-b1 is one of factors in the dentin organic matrix playing at least a partial role in osteoclastogenesis. The knowledge gained from this study may help us to understand the factors that influence the mechanisms of resorption, leading to the development of new therapies for the treatment of hard tissue diseases. However, further experiments are still needed to determine other possible factors involved in osteoclast formation. Acknowledgments This study was supported by a Grant-in-Aid for Scientific Research (20592392) from the Ministry of Education, Culture, Sports, Science and Technology, Japan and the Chulalongkorn University Centenary Academic Development Project (to WS), Thailand. Conflict of interest of interest.

The authors declare that they have no conflict

References 1. Boyde A, Ali NN, Jones SJ. Resorption of dentine by isolated osteoclasts in vitro. Br Dent J. 1984;156(6):216–20. 2. Sasaki T. Differentiation and functions of osteoclasts and odontoclasts in mineralized tissue resorption. Microsc Res Tech. 2003;61(6):483–95. 3. Sasaki T, Motegi N, Suzuki H, Watanabe C, Tadokoro K, Yanagisawa T, et al. Dentin resorption mediated by odontoclasts in physiological root resorption of human deciduous teeth. Am J Anat. 1988;183(4):303–15.

Odontology 4. Butler WT, Ritchie H. The nature and functional significance of dentin extracellular matrix proteins. Int J Dev Biol. 1995;39(1):169–79. 5. Veis A. Dentin composition. In: Lazzari E, editor. Handbook of experimental aspects of oral biochemistry. Florida: CRC Press, Inc; 1983. p. 71–83. 6. Ritchie HH, Hou H, Veis A, Butler WT. Cloning and sequence determination of rat dentin sialoprotein, a novel dentin protein. J Biol Chem. 1994;269(5):3698–702. 7. MacDougall M, Zeichner-David M, Slavkin HC. Production and characterization of antibodies against murine dentine phosphoprotein. Biochem J. 1985;232(2):493–500. 8. Finkelman RD, Mohan S, Jennings JC, Taylor AK, Jepsen S, Baylink DJ. Quantitation of growth factors IGF-I, SGF/IGF-II, and TGF-beta in human dentin. J Bone Miner Res. 1990; 5(7):717–23. 9. Russo LG, Maharajan P, Maharajan V. Basic fibroblast growth factor (FGF-2) in mouse tooth morphogenesis. Growth Factors (Chur, Switzerland). 1998;15(2):125–33. 10. Thomadakis G, Ramoshebi LN, Crooks J, Rueger DC, Ripamonti U. Immunolocalization of bone morphogenetic protein-2 and -3 and osteogenic protein-1 during murine tooth root morphogenesis and in other craniofacial structures. Eur J Oral Sci. 1999; 107(5):368–77. 11. Nesbitt SA, Horton MA. Trafficking of matrix collagens through bone-resorbing osteoclasts. Science. 1997;276(5310):266–9. 12. Lara VS, Figueiredo F, da Silva TA, Cunha FQ. Dentin-induced in vivo inflammatory response and in vitro activation of murine macrophages. J Dent Res. 2003;82(6):460–5. 13. Ogata Y, Niisato N, Moriwaki K, Yokota Y, Furuyama S, Sugiya H. Cementum, root dentin and bone extracts stimulate chemotactic behavior in cells from periodontal tissue. Comp Biochem Physiol B: Biochem Mol Biol. 1997;116(3):359–65. 14. Chellaiah MA, Kizer N, Biswas R, Alvarez U, Strauss-Schoenberger J, Rifas L, et al. Osteopontin deficiency produces osteoclast dysfunction due to reduced CD44 surface expression. Mol Biol Cell. 2003;14(1):173–89. 15. Silva TA, Lara VS, Silva JS, Garlet GP, Butler WT, Cunha FQ. Dentin sialoprotein and phosphoprotein induce neutrophil recruitment: a mechanism dependent on IL-1beta, TNF-beta, and CXC chemokines. Calcif Tissue Int. 2004;74(6):532–41. 16. Silva TA, Lara VS, Silva JS, Oliveira SH, Butler WT, Cunha FQ. Macrophages and mast cells control the neutrophil migration induced by dentin proteins. J Dent Res. 2005;84(1):79–83. 17. Kaneko H, Arakawa T, Mano H, Kaneda T, Ogasawara A, Nakagawa M, et al. Direct stimulation of osteoclastic bone resorption by bone morphogenetic protein (BMP)-2 and expression of BMP receptors in mature osteoclasts. Bone. 2000;27(4):479–86. 18. Wang Y, Nishida S, Elalieh HZ, Long RK, Halloran BP, Bikle DD. Role of IGF-I signaling in regulating osteoclastogenesis. J Bone Miner Res. 2006;21(9):1350–8. 19. Kawaguchi H, Chikazu D, Nakamura K, Kumegawa M, Hakeda Y. Direct and indirect actions of fibroblast growth factor 2 on osteoclastic bone resorption in cultures. J Bone Miner Res. 2000;15(3):466–73. 20. D’Souza RN, Flanders K, Butler WT. Colocalization of TGF-beta 1 and extracellular matrix proteins during rat tooth development. Proc Finn Dent Soc. 1992;88(Suppl 1):419–26. 21. Vaahtokari A, Vainio S, Thesleff I. Associations between transforming growth factor beta 1 RNA expression and epithelialmesenchymal interactions during tooth morphogenesis. Development. 1991;113(3):985–94. 22. Cassidy N, Fahey M, Prime SS, Smith AJ. Comparative analysis of transforming growth factor-beta isoforms 1–3 in human and rabbit dentine matrices. Arch Oral Biol. 1997;42(3):219–23.

23. Magloire H, Romeas A, Melin M, Couble ML, Bleicher F, Farges JC. Molecular regulation of odontoblast activity under dentin injury. Adv Dent Res. 2001;15:46–50. 24. Melin M, Joffre-Romeas A, Farges JC, Couble ML, Magloire H, Bleicher F. Effects of TGFbeta1 on dental pulp cells in cultured human tooth slices. J Dent Res. 2000;79(9):1689–96. 25. DenBesten PK, Machule D, Gallagher R, Marshall GW Jr, Mathews C, Filvaroff E. The effect of TGF-beta 2 on dentin apposition and hardness in transgenic mice. Adv Dent Res. 2001;15:39–41. 26. Hattersley G, Chambers TJ. Effects of transforming growth factor beta 1 on the regulation of osteoclastic development and function. J Bone Miner Res. 1991;6(2):165–72. 27. Fuller K, Lean JM, Bayley KE, Wani MR, Chambers TJ. A role for TGFbeta(1) in osteoclast differentiation and survival. J Cell Sci. 2000;113(Pt 13):2445–53. 28. Karsdal MA, Hjorth P, Henriksen K, Kirkegaard T, Nielsen KL, Lou H, et al. Transforming growth factor-beta controls human osteoclastogenesis through the p38 MAPK and regulation of RANK expression. J Biol Chem. 2003;278(45):44975–87. 29. Quinn JM, Itoh K, Udagawa N, Hausler K, Yasuda H, Shima N, et al. Transforming growth factor beta affects osteoclast differentiation via direct and indirect actions. J Bone Miner Res. 2001;16(10):1787–94. 30. Sriarj W, Aoki K, Ohya K, Takagi Y, Shimokawa H. Bovine dentine organic matrix down-regulates osteoclast activity. J Bone Miner Metab. 2009;27(3):315–23. 31. Takagi Y, Veis A. Isolation of phosphophoryn from human dentin organic matrix. Calcif Tissue Int. 1984;36(3):259–65. 32. Grcevic D, Lukic IK, Kovacic N, Ivcevic S, Katavic V, Marusic A. Activated T lymphocytes suppress osteoclastogenesis by diverting early monocyte/macrophage progenitor lineage commitment towards dendritic cell differentiation through downregulation of receptor activator of nuclear factor-kappaB and c-Fos. Clin Exp Immunol. 2006;146(1):146–58. 33. Akatsu T, Tamura T, Takahashi N, Udagawa N, Tanaka S, Sasaki T, et al. Preparation and characterization of a mouse osteoclastlike multinucleated cell population. J Bone Miner Res. 1992;7(11):1297–306. 34. Nakasato YR, Janckila AJ, Halleen JM, Vaananen HK, Walton SP, Yam LT. Clinical significance of immunoassays for type-5 tartrateresistant acid phosphatase. Clin Chem. 1999;45(12):2150–7. 35. Maeda S, Hayashi M, Komiya S, Imamura T, Miyazono K. Endogenous TGF-beta signaling suppresses maturation of osteoblastic mesenchymal cells. EMBO J. 2004;23(3):552–63. 36. Shen L, Smith JM, Shen Z, Eriksson M, Sentman C, Wira CR. Inhibition of human neutrophil degranulation by transforming growth factor-beta1. Clin Exp Immunol. 2007;149(1):155–61. 37. Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell. 1998;93(2):165–76. 38. Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA. 1998;95(7):3597–602. 39. Li J, Sarosi I, Yan XQ, Morony S, Capparelli C, Tan HL, et al. RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. Proc Natl Acad Sci USA. 2000;97(4):1566–71. 40. Yan T, Riggs BL, Boyle WJ, Khosla S. Regulation of osteoclastogenesis and RANK expression by TGF-beta1. J Cell Biochem. 2001;83(2):320–5. 41. Koseki T, Gao Y, Okahashi N, Murase Y, Tsujisawa T, Sato T, et al. Role of TGF-beta family in osteoclastogenesis induced by RANKL. Cell Signal. 2002;14(1):31–6.

123

Odontology 42. Jensen ED, Pham L, Billington CJ Jr, Espe K, Carlson AE, Westendorf JJ, et al. Bone morphogenic protein 2 directly enhances differentiation of murine osteoclast precursors. J Cell Biochem. 2010;109(4):672–82. 43. Cowan CM, Aalami OO, Shi YY, Chou YF, Mari C, Thomas R, et al. Bone morphogenetic protein 2 and retinoic acid accelerate in vivo bone formation, osteoclast recruitment, and bone turnover. Tissue Eng. 2005;11(3–4):645–58.

123

44. Yasui T, Kadono Y, Nakamura M, Oshima Y, Matsumoto T, Masuda H, et al. Regulation of RANKL-induced osteoclastogenesis by TGF-beta through molecular interaction between Smad3 and Traf6. J Bone Miner Res. 2011;26(7):1447–56. 45. Fox SW, Evans KE, Lovibond AC. Transforming growth factorbeta enables NFATc1 expression during osteoclastogenesis. Biochem Biophys Res Commun. 2008;366(1):123–8.